Schwann cells originating in myeloid interstitial cells

ABSTRACT

There is provided a method of inducing bone marrow stromal cells to differentiate into bone marrow stromal cell-derived Schwann cells in vitro, comprising the steps of: collecting bone marrow stromal cells from bone marrow and culturing the cells in a standard essential culture medium supplemented with a serum; adding a reducing agent to the culture medium and further culturing the cells; adding a differentiation inducing agent to the culture medium and further culturing the cells; and adding a cyclic AMP-augmenting agent or a cyclic AMP analogue and/or a glial cell differentiation and survival stimulating factor to the culture medium, and further culturing the cells to obtain the bone marrow stromal cell-derived Schwann cells. There are also provided bone marrow stromal cell-derived Schwann cells obtained thereby and a pharmaceutical composition for neural regeneration that comprises them.

TECHNICAL FIELD

[0001] The present invention relates to an in vitro method of inducingbone marrow stromal cells to differentiate into Schwann cells, to thebone marrow stromal cell-derived Schwann cells, to a pharmaceuticalcomposition for neural regeneration that comprises them, and to a methodfor treatment of neural diseases using the Schwann cells and thecomposition.

BACKGROUND ART

[0002] Damage to the nervous system, and particularly the centralnervous system including brain, spinal cord, and optic nerve is believedto be irreversible, leading ultimately to the process of degeneration.Traffic accidents and sports injuries, ischemia, tumors, prolongedinflammation, cryptogenic degenerative disease and the like are amongthe causes of neurological diseases which occur with a high incidenceamong the population, and are of urgent social significance.

[0003] The irreversibility of central nerve damage is attributed to theglial environment of nerve tissue. The brain and spinal cord have thesame glial environment, which will now be described using the opticnerve as an example of a central nerve.

[0004] 1) First, nerve fibers undergo degeneration and graduallydisappear. During the process, the myelin sheaths covering the nervesalso degenerate leaving cell residues (see FIG. 1b). The myelin sheathsformed by oligodendrocytes contain substances which strongly inhibitnerve fiber regeneration and elongation⁽¹⁾.

[0005] 2) The astrocytes proliferate and enlarge, resulting in gliosis(see FIG. 1b). More specifically, they displace the nerve fibers,occupying the primary locations and thereby physically inhibitingregeneration⁽²⁾. The astrocytes forming the gliosis exhibit a morphologycontrasting considerably with that of normal astrocytes, with a greaternumber of processes and intricately complex forms. Particularly in thecase of injury, the site of damage shows numerous layers of astrocytesstacked orthogonally to the direction of extension of the nerve fibersand linked together at their processes, forming a cap-like barrierstructure.

[0006] 3) Processing of the oligodendrocytes and their cell residuesubstances such as myelin after degeneration is slower compared to otherregenerating tissues such as peripheral nerve system. The main reasonfor this is presumably the very low degree of infiltration of peripheralimmune cells such as macrophages and monocytes, resulting in delayedprocessing of the residue in the early stages.

[0007] The following explanations have been proposed for lack ofregeneration of optic nerves.

[0008] 1) As mentioned above, oligodendrocytes have a strong inhibitingeffect on neural regeneration. Specifically, the molecule Nogo extractedfrom oligodendrocytes has been shown to be an inhibitor^((1,9)).Experiments with culturing systems have shown that during extension ofneurites, the processes contacting with the myelin sheaths ofoligodendrocytes not only stop extending, but even regress (contactinhibition). Moreover, neurites do not extend at myelinated areas, andin culturing the processes grow to avoid them.

[0009] 2) Gliotic astrocytes produce various inhibiting substances,including proteoglycans such as keratin (3) sulfate and chondroitinsulfate⁽³⁾.

[0010] 3) The optic nerves and the entire central nervous system areexceedingly silent even after suffering injury. The system is not underimmune surveillance, and this is instead considered to be a disadvantagefor regeneration. For example, the peripheral nerve system describedhereunder differs from the optic nerve even in the structure of theglia, and it has been compared and studied as a regenerating system eventhough it is composed of the same nerve tissue. Upon injury ofperipheral nerve tissue, immune cells such as peripheral macrophagesrapidly infiltrate (within a few hours to a couple of days) to processthe cell residue. Meanwhile, cytokines are secreted in large amounts,promoting regeneration of the nerve tissue. Such neural regenerationoccurs in concert with a cascade of phenomena with one reaction leadingto another, but no infiltration of macrophages is seen in the opticnerve in the early stages. This has been attributed to the suppressionof macrophage activity in the central nervous system including the opticnerve⁽⁴⁾. It appears that this suppressing function arose in order toprevent macrophage digestion of complex developed central neural nets.It has therefore been conjectured that it is the absence of the firsttrigger in the optic nerves that leads to degeneration instead ofregeneration.

[0011] 4) In order for regeneration to occur, a structural scaffoldingis necessary to induce nerve fibers. However, once degeneration hasoccurred in the optic nerves, the route for regeneration is lost. In theoptic nerves, each oligodendrocyte forms numerous myelin sheaths on thenerve fibers, and astrocytes surround the myelinated fibers and coverthe unmyelinated fiber bundles (see FIG. 2, bottom). The basal membraneis present only outside of the astrocytes forming the membrana limitansglia, or in other words, the entire optic nerve may be considered to beinside a single basal membrane sheath. In the peripheral nerves, thebasal membrane serves as a route for regeneration. Consequently, oncethe nerve fibers in the optic nerve have degenerated, the route whichonce existed for each of the nerve fibers is no longer present.

[0012] Yet peripheral nerves, unlike central nerves, are capable ofregeneration.

[0013] Unlike central nerves, the primary cells of the peripheral nervesare Schwann cells. All of the nerve fibers, whether myelinated orunmyelinated, are covered with Schwann cells (see FIG. 2, top). Schwanncells are derived from neural crest cells, while the central glia(oligodendrocytes, astrocytes) which act inhibitorily on neuralregeneration are derived from neural tubes, and therefore the sources ofdifferentiation are different.

[0014] Peripheral nerves are believed to regenerate in the followingfashion.

[0015] 1) When injury such as a cut is suffered by peripheral nerves,Wallerian degeneration occurs at the peripheral end from the site ofinjury (see FIG. 3). The Schwann cells return to an undifferentiatedstate from the myelin-forming differentiated type, and are thenactivated to divide and proliferate, exhibiting a funicular form. In theperipheral nerves, the individual nerve fibers are independentlysurrounded by Schwann cells, with the outer area covered by a basalmembrane (see FIG. 2). That is, each of the nerves resides within aseparate basal membrane sheath. Thus even when degeneration occurs, theSchwann cells activated in the basal membrane sheath proliferate to forma funicular structure, thus providing a foothold for reconstruction ofthe neural network. Wallerian degeneration, therefore, is notdegeneration in the strict sense but rather the first step towardregeneration.

[0016] 2) Peripheral macrophages play a major role in the process ofWallerian degeneration. The macrophages infiltrate rapidly at theperipheral end of the site of injury, processing the remnants of thedegenerated nerves fibers and myelin (see FIG. 3) while also secretingcytokines such as IL-1 to activate the Schwann cells. Although nodefinite conclusions can be drawn regarding the cause which inducesfiltration of macrophages, the Schwann cells themselves have beenindicated as a likely candidate. In any case, it is believed that theSchwann cells activated by the macrophage signals synthesize variousfactors indispensable for regeneration, such as nerve growth factorwhich will be explained below, and guide regeneration of the nerve.

[0017] 3) The myelin sheaths of Schwann cells have a low inhibitoryeffect. This is a major difference from the myelin of oligodendrocyteswhich does exhibit an inhibitory effect. In addition, it is known thatthe composition of the myelin protein of Schwann cells andoligodendrocytes differs.

[0018] Thus, even when injured, the central glia do not revert back totheir differentiated state to undergo differentiation and proliferationor significantly alter their form, as do the Schwann cells of theperipheral nerves, but instead maintain their relatively differentiatedphenotype. The peripheral nerves on the other hand are characterized byexhibiting a rapid, highly flexible response to injury.

[0019] Schwann cells are considered to play the following role in neuralregeneration.

[0020] Schwann cells produce numerous factors and secrete them in adiffuse manner. Moreover, their own cell membrane surfaces are coveredwith a basal membrane, and extracellular matrix components are includedtherein. Cell adhesion molecules are also known to be expressed inSchwann cell membranes, and it is thought that these factors act as awhole to induce neural regeneration (see FIG. 4).

[0021] 1. Secreted Factors

[0022] Schwann cells are known to produce many neurotrophic factors,among which the following are the main ones involved in regeneration: 1)the neurotrophin family, including nerve growth factor, brain-derivedneurotrophic factor, neurotrophin-3, neurotrophin-4/5; 2) ciliaryneurotrophic factor; 3) the FGF family, including acidic and basicfibroblast growth factor; 4) the insulin family, including insulin-likegrowth factor-I and II; and 5) transforming growth fator-β2 and β3.

[0023] These neurotrophins have powerful effects not only on thesurvival of neurons but also on neurite elongation and the like. Otherfactors also have neurotrophic effects and neurite elongating effects onnerves, but their action mechanisms are considered to be autocrinicsince they simultaneously activate the Schwann cells themselves.

[0024] 2. Extracellular Matrix Components

[0025] These include fibronectin, laminin, type IV collagen andtenascin. Based on experiments with cultured systems, it is believedthat fibronectin and laminin play a supporting role in neuralregeneration.

[0026] 3. Cell Adhesion Molecules

[0027] A large number of cell adhesion molecules have been identified.The following description will focus on those associated with Schwanncells and neural regeneration.

[0028] 1) Immunoglobulin Superfamily

[0029] NCAM (Neural Cell Adhesion Molecule) and L1 are expressed onSchwann cell membranes and play important roles as adhesion moleculesduring elongation of the nerve fibers as they contact with the Schwanncell scaffolding. Both are connected to the cytoskeleton and function tomaintain the shape of the cell, while also accomplishing intracellularactivation through inositol phosphate system and calcium channelactivation. In addition, MAG (Myelin Associated Glycoprotein) isexpressed between Schwann cells after nerve elongation has progressed tosome degree and remyelination of the nerve fibers has begun.

[0030] 2) Cadherin Superfamily

[0031] Cadherins are calcium-dependent cell adhesion molecules of whichnumerous types have been identified. N-cadherin is associatedparticularly with neural regeneration. Like NCAM and L1 of theimmunoglobulin superfamily, this molecule also plays an important roleduring elongation of the nerve fibers as they contact with and recognizeSchwann cells.

[0032] 3) Integrin Superfamily

[0033] Integrins are cellular receptors for the aforementionedextracellular matrix components. They are heterodimeric moleculescomposed of two subunits, α and β. Like cadherins, they are also thoughtto link with the cytoskeleton and function directly in signaltransduction between cells. Schwann cells express the α₆β₄ subtype whichplays a role in the process of remyelination during regeneration.

[0034] Regeneration of central nerves has been attempted in the pastseveral decades or so by numerous researchers. The following is asummary of those attempts.

[0035] 1) Regeneration has been achieved by cutting portions ofperipheral nerves of the hand or foot and autografting them into thecentral nerves. Thus, peripheral nerves presumably possess anenvironment which promotes regeneration of central nerves. Such studiesbegan with research by Aguayo et al. in Canada in 1983⁽⁸⁾.

[0036] 2) As mentioned above, the central nerves themselves act tosuppress neural regeneration. Reports have been published on theinhibiting effects of previously known myelin-related proteins,including Nogo factor which was described in 2000 in the journalNature⁽⁹⁾. According to several reports, introducing antibodies againstthese factors to neutralize them can induce some degree of regenerationin the central nerves.

[0037] 3) Regeneration is promoted by the following two types of celltransplantation.

[0038] a) Replenishment of degenerated neurons to reconstruct the neuralnetwork. This approach employs neuronal stem cells and embryonicneurons.

[0039] b) Reconstruction by transplantation of cells capable of inducingneural regeneration (glial cells), instead of replenishing the actualneurons. It has been attempted to use peripheral nerve-derived Schwanncells, central nerve-derived glial cells or central nerve-derivedependymal cells, having neurotrophic factors introduced, olfactorynerve-derived support cells, neuronal stem cells and the like.

[0040] Both methods have advantages and disadvantages, but as yet norevolutionary method has been developed.

[0041] The present inventors have for many years been involved indevelopment of methods of neural regeneration and reestablishment offunction. We have focused particularly on a method employing Schwanncells which support the tissue structure of peripheral nerves asdescribed in 3)-b) above. Schwann cells are present in peripheralnerves, and it has been demonstrated that they are capable of inducingregeneration not only of their own peripheral nerve tissue but also ofthe central nervous system, that their transplantation at sites ofinjury provides a foothold for regenerating fibers and leads toeffective neural regeneration, and that myelin which is responsible forneural saltatory transmission as an indispensable element for normalnerve functioning can also be reconstructed by transplantation ofSchwann cells. It has also been confirmed in animal experiments thattransplantation of Schwann cells leads to regeneration of cut opticnerves, (central nervous system).

[0042] Nevertheless, various difficulties are encountered when therelatively simple procedure of collecting and culturing Schwann cells inanimal experiments is applied to humans. For example, since Schwanncells are present in the peripheral nerves, it is necessary to extractnerve samples from the hands or feet and isolate the cells, therebyleaving damage in the donor after extraction. As an additionaldifficulty, the limited proliferating ability of adult-derived Schwanncells requires a greater time period for large-scale culturing.Moreover, neural crest cells, which are believed to differentiate intoSchwann cells, can only be extracted from embryonic peripheral nerves.

[0043] This situation has therefore necessitated provision of a naturalSchwann cell substitute which can be used for neural regenerationtreatment and can be obtained in large amounts by culturing.

[0044] Neuronal stem cells have been found in portions of the adultbrain, and these differentiate into the neurons, astrocytes,oligodendrocytes, etc. of the nervous system (see FIG. 5). However, onlya very minute number of such stem cells are present, and craniotomy isnecessary to obtain them. In addition, recent research has shown,contrary to germ layer theory, that some types of cells may be able todifferentiate into completely different types (see FIG. 6). As of thefiling of the present application it has been known that bone marrowstromal cells are mesenchymal stem cells or precursor cells that notonly carry out a hemopoietic support function but can themselvesdifferentiate into osteoblasts, vascular endothelial cells, skeletalmuscle cells, adipocytes, smooth muscle cells and the like⁽¹⁰⁾;nevertheless, no literature exists suggesting the possibility that bonemarrow stromal cells might be capable of differentiating into neuralcrest cell-derived Schwann cells, nor has any method for suchdifferentiation or induction been established.

[0045] In light of this situation, the present inventors have attemptedexperimentation and research on differentiation and induction to Schwanncells using bone marrow stromal cells instead of neural crest cells thatare so difficult to obtain, as mentioned above. Bone marrow stromalcells are easy to extract by bone marrow puncture on an outpatient basisand have high proliferation potency, and thus allow large-scaleculturing in a relatively short period of time.

DISCLOSURE OF THE INVENTION

[0046] As a result of repeated experimentation, the present inventorsare the first to have succeeded in inducing differentiation of bonemarrow stromal cells into Schwann cells with a high degree of efficiencyby a multistage operation. Moreover, it was confirmed that actualregeneration and elongation of nerves occurred upon transplanting thebone marrow stromal cell-derived Schwann cells obtained by thedifferentiation inducing method into damaged optic nerves (centralnervous system), and the present invention was thus completed.

[0047] The present invention therefore provides a method of inducingbone marrow stromal cells to differentiate into bone marrow stromalcell-derived Schwann cells in vitro, comprising the steps of:

[0048] (1) collecting bone marrow stromal cells from bone marrow andculturing the cells in a standard essential culture medium supplementedwith a serum;

[0049] (2) adding a reducing agent to the culture medium and furtherculturing the cells;

[0050] (3) adding a differentiation inducing agent to the culture mediumand further culturing the cells; and

[0051] (4) adding a cyclic AMP-augmenting agent or a cyclic AMP analogueand/or a glial cell differentiation and survival stimulating factor tothe culture medium, and further culturing the cells to obtain the bonemarrow stromal cell-derived Schwann cells.

[0052] The invention further provides bone marrow stromal cell-derivedSchwann cells obtained by the aforementioned method and a pharmaceuticalcomposition for neural regeneration comprising the bone marrow stromalcell-derived Schwann cells. The invention still further provides amethod of treating neural disease by transplanting the aforementionedbone marrow stromal cell-derived Schwann cells or a pharmaceuticalcomposition for neural regeneration comprising them into a patient withneural disease to promote regeneration of the neural cells with whichthe disease is associated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053]FIG. 1 is an illustration of the changes which occur in opticnerve glial cells after injury. Here, FIG. 1a shows the state of anormal optic nerve, and FIG. 1b shows the state of an injured opticnerve.

[0054]FIG. 2 is an illustration of the structural differences betweenperipheral nerves and central nerves (including optic nerves).

[0055]FIG. 3 is an illustration of Wallerian degeneration of aperipheral nerve. The normal nerve fiber is ensheathed by Schwann cells,and the exterior further covered by a continuous basal membrane (FIG.3a). Upon injury, the peripheral end of the nerve fiber degenerates intomyelin remnants which are processed by approaching macrophages (FIG.3b), and after processing of the remains, activated Schwann cellsproliferate inside the tube of the remaining basal membrane (FIG. 3c),leading to remyelination to complete the regenerated nerve fiber (FIG.3d). (“Peripheral Nerve Injury and Repair”, translation supervised by Y.Ikuta, Yodogawa Publications, FIG. 5.1, revised 1991.)

[0056]FIG. 4 is an graphical listing of neurotrophic factors associatedwith Schwann cells. Schwann cells produce secreted factors,extracellular matrix components and cell adhesion molecules, which workin concert for regeneration.

[0057]FIG. 5 is a schematic diagram for differentiation of neural cells.

[0058]FIG. 6 is an illustration of differentiation whereby, contrary togerm layer theory, certain cells may be induced into completelydifferent cell types.

[0059]FIG. 7 is a composite of immunofluorescent photographs in lieu ofa drawing, showing the features of bone marrow stromal cells beforedifferentiation has been induced.

[0060]FIG. 8 is a composite of immunofluorescent photographs in lieu ofa drawing, showing the features of bone marrow stromal cell-derivedSchwann cells compared to natural Schwann cells.

[0061]FIG. 9 is a composite of micrographs (phase contrast micrograph)in lieu of a drawing, showing the features of bone marrow stromalcell-derived Schwann cells obtained by the differentiation inducingmethod of the invention, compared to natural Schwann cells.

[0062]FIG. 10 is a composite of micrographs (phase contrast micrograph)in lieu of a drawing, showing the morphology of bone marrow stromalcell-derived Schwann cells obtained by the differentiation inducingmethod of the invention and cells obtained by the same method with someof the steps omitted, compared to natural Schwann cells and natural bonemarrow stromal cells.

[0063]FIG. 11 is a flow chart summary of the differentiation inducingmethod of the invention.

[0064]FIG. 12 is a pair of immunohistological confocal laser micrographsin lieu of a drawing, showing regeneration of an optic nerve aftertransplantation of bone marrow stromal cell-derived Schwann cells, usingGAP43 as the indicator.

[0065]FIG. 13 is a pair of immunohistological confocal laser micrographsin lieu of a drawing, showing regeneration of an optic nerve aftertransplantation of bone marrow stromal cell-derived Schwann cells, usingFITC, TexRed and Alexa633 as indicators.

[0066]FIG. 14 is an immunohistological confocal laser micrograph in lieuof a drawing, showing regeneration of a sciatic nerve aftertransplantation of bone marrow stromal cell-derived Schwann cells, usingGAP43 as the indicator.

[0067]FIG. 15 is a set of immunohistological confocal laser micrographsin lieu of a drawing, showing regeneration of sciatic nerve aftertransplantation of bone marrow stromal cell-derived Schwann cells, usingGFP, neurofilament and MAG.

[0068]FIG. 16 is a pair of immunohistological confocal laser micrographsin lieu of a drawing, showing regeneration of sciatic nerve aftertransplantation of bone marrow stromal cell-derived Schwann cells, usingGFP, neurofilament and MAG.

[0069]FIG. 17 is another pair of immunohistological confocal lasermicrographs in lieu of a drawing, showing regeneration of sciatic nerveafter transplantation of bone marrow stromal cell-derived Schwann cells,using GFP, neurofilament and MAG.

[0070]FIG. 18 is another pair of immunohistological confocal lasermicrographs in lieu of a drawing, showing regeneration of sciatic nerveafter transplantation of bone marrow stromal cell-derived Schwann cells,using GFP, neurofilament and MAG.

DETAILED DESCRIPTION OF THE INVENTION

[0071] According to one mode of the present invention there is provideda method of inducing bone marrow stromal cells to differentiate intobone marrow stromal cell-derived Schwann cells in vitro, comprising thesteps of:

[0072] (1) collecting bone marrow stromal cells from bone marrow andculturing the cells in a standard essential culture medium supplementedwith a serum;

[0073] (2) adding a reducing agent to the culture medium and furtherculturing the cells;

[0074] (3) adding a differentiation inducing agent to the culture mediumand further culturing the cells; and

[0075] (4) adding a cyclic AMP-augmenting agent or a cyclic AMP analogueand/or a glial cell differentiation and survival stimulating factor tothe culture medium, and further culturing the cells to obtain the bonemarrow stromal cell-derived Schwann cells.

[0076] The density of the cells in step (1) may be 50% confluency, andthe cells are preferably subcultured to four generations.

[0077] The standard essential culture medium may be Minimum EssentialMedium Eagle Alpha Modification (M4526, Sigma) and the serum may befetal calf serum (14-501F, Lot #61-1012, BioWhittaker Co.). The serummay be added to a concentration of 20%. The reducing agent is an SHreagent, and the SH reagent is preferably β-mercaptoethanol (214-18,Lot# MOM7582, Nacalai Tesque). The concentration of the reducing agentmay be 1 nM to 10 mM, preferably 10 nM to 5 mM and more preferably 100μM to 2 mM. The culturing time in step (2) may be 1 hour to 5 days,preferably 12-48 hours and more preferably 18-30 hours. Theaforementioned reagent concentration is the concentration in the culturemedium with which the cells are in direct contact (same for reagentsreferred to hereunder).

[0078] The differentiation inducing agent may be retinoic acid(all-trans) (R-2625, Sigma). The differentiation inducing agentconcentration may be 0.001 ng/ml to 1 μg/ml, preferably 1 ng/ml to 200ng/ml and more preferably 10 ng/ml to 60 ng/ml. In step (3), the culturemedium used in step (2) may be exchanged with fresh differentiationinducing agent-containing medium after step (2) has been completed. Thefresh culture medium is identical to the culture medium used in step (1)except that it contains the differentiation inducing agent. Theculturing time for step (3) may be 1 hour to 30 days, preferably 12hours to 7 days and more preferably 2-4 days.

[0079] The cyclic AMP-augmenting agent or cyclic AMP analogue may beforskolin (344273, Calbiochem). The concentration of the cyclicAMP-augmenting agent or cyclic AMP analogue may be 0.001 ng/ml to 100μg/ml, preferably 100 ng/ml to 50 μg/ml and more preferably 1 μg/ml to10 μg/ml.

[0080] The glial cell differentiation and survival stimulating factormay be one selected from the group consisting of neuregulin,platelet-derived growth factor-AA (396-HB, Peprotech EC, Ltd.), basicfibroblast growth factor (100-18B, Peprotech EC, Ltd.) or mixturesthereof. Neuregulin is available as Heregulin™ (396-HB, R&D Corp.) Theconcentration of the glial cell differentiation and survival stimulatingfactor may be 0.001 ng/ml to 100 μg/ml, with a concentration ofpreferably 0.1 ng/ml to 100 ng/ml and more preferably 1 ng/ml to 10ng/ml for platelet-derived growth factor-AA, and a concentration ofpreferably 10 ng/ml to 1 μg/ml and more preferably 100 ng/ml to 300ng/ml for basic fibroblast growth factor. The culturing time in step (4)may be 1 hour to 30 days, and preferably 4 to 10 days.

[0081] According to another mode of the invention there are providedbone marrow stromal cell-derived Schwann cells obtained by theaforementioned differentiation inducing method.

[0082] According to yet another mode of the invention there is provideda pharmaceutical composition for neural regeneration comprising bonemarrow stromal cell-derived Schwann cells.

[0083] According to yet another mode of the invention there is provideda method of treating neural disease by transplanting the aforementionedbone marrow stromal cell-derived Schwann cells or a pharmaceuticalcomposition for neural regeneration comprising them into a patient withneural disease to cause regeneration of the neural cells with which thedisease is associated.

[0084] Throughout the present specification, the term “bone marrowstromal cells” refers to cells in the bone marrow which are not of thehemopoietic system and are considered capable of differentiating tocells of the bone, cartilage, etc. Bone marrow stromal cells arepositive for Thy1.2 and (β1-integrin) and negative for CD34, as shown inthe immunofluorescent photographs of FIG. 7. They may be positive ornegative for S-100 (calcium-binding protein). Antibodies for Thy1.2,β1-integrin and CD34 were used.

[0085] Throughout the present specification, the term “natural Schwanncells” refers to Schwann cells collected from the peripheral nerves ofliving bodies, namely dorsal root ganglions. As seen in the upperimmunofluorescent photograph of FIG. 8, they are positive for S-100.

[0086] Throughout the present specification, the term “bone marrowstromal cell-derived Schwann cells” refers to Schwann cells which (1)closely resemble natural Schwann cells in morphology and do not revertto the form of bone marrow stromal cells by subculturing, (2) exhibitthe same reaction as Schwann cells with respect to P75 (nerve growthfactor (NGF) receptor, low affinity), S-100, GFAP (glial fibrillaryacidic protein, a type of intermediate filament), nestin (a type ofintermediate filament) and O4 (a marker for myelin-producing cells suchas Schwann cells and oligodendrocytes) based on immunostaining as shownin the lower immunofluorescent photographs of FIG. 8, and (3) havefeatures similar to natural Schwann cells in their neurogenic ability,but can be distinguished from natural Schwann cells due to theirdistinct differentiation histories. Antibodies for P75, S-100, GFAP,nestin and O4 were obtained from the following sources: Anti-nervegrowth factor-receptor, Boehringer Mannheim, 1198645; Anti-S-100, z-311,Dako Corp.; Anti-glial fibrillary acidic protein, L-1812, Dako Corp.;Anti-nestin, BMS4353, Bioproducts; Anti-O4, 1518925, BoehringerMannheim.

[0087] Bone marrow was treated in multistages according to theinvention, as shown in the micrograph of FIG. 9.

[0088] Stromal cells (right upper and lower photographs in FIG. 9)exhibit the same morphology as natural Schwann cells (left photograph inFIG. 9).

[0089]FIG. 10 shows the morphology of bone marrow stromal cells obtainedby the method of inducing differentiation of bone marrow stromal cellsaccording to the invention, without steps (2) to (4) or some of thereagents used therein. The micrograph at the upper left in FIG. 10 showsnatural Schwann cells. The micrograph at the lower right shows bonemarrow stromal cells before treatment. The top center micrograph showsresults by the method of inducing differentiation of bone marrow stromalcells according to the invention conducted without omission of steps (2)to (4), and it is seen that the method of the invention produced cellsexhibiting a morphology similar to natural Schwann cells. The micrographat the upper right was obtained with omission of step (4), and themicrographs at the lower left and bottom center were taken without thedifferentiation inducing agent retinoic acid and without forskolin instep (3) and step (4), respectively.

[0090] It was thus demonstrated that the multistage treatment describedin steps (2) to (4) above induces differentiation of bone marrow stromalcells into bone marrow stromal cell-derived Schwann cells with highefficiency.

[0091] The term “high efficiency” as used throughout the presentspecification means that the differentiation inducing method of theinvention converts a high proportion of the original bone marrow stromalcells into the final bone marrow stromal cell-derived Schwann cells. Thehigh efficiency of the differentiation inducing method of the inventionis 50% or greater, preferably 75% or greater, more preferably 90% orgreater and most preferably 95% or greater. Although each of theindividual steps described above have been known, the selection andoptimum combination of the steps as according to the present inventionwere first discovered by the present inventors, and the discovery ishighly significant. Specifically, while it was known that bone marrowstromal cells are mesenchymal stem cells or precursor cells that arecapable of being induced to differentiate into osteoblasts, vascularendothelial cells, skeletal muscle cells, adipocytes, smooth musclecells and the like, as explained above, it was not known whether bonemarrow stromal cells could actually be differentiated into neural crestcell-derived Schwann cells, and no party had successfully achieved thisdespite a strong desire to do so. The present inventors, though notwishing to be constrained by any theory, conjecture that the treatmentwith a reducing agent in step (2) produces a shock on the cells whilethe treatment with retinoic acid in step (3) resets the cells, afterwhich the treatment with a cyclic AMP-augmenting agent or cyclic AMPanalogue and a glial cell differentiation and survival stimulatingfactor in step (4) induces differentiation of the cells.

[0092] Bone marrow stromal cells may be collected and subjected totreatment involving multiple steps according to the present invention,to induce their differentiation into cells having the same features asnatural Schwann cells with respect to neurogenic ability, with highefficiency. By transplanting the bone marrow stromal cell-derivedSchwann cells into the peripheral and central nervous system, it hasbecome possible to induce regeneration and elongation of injured nerves.

[0093] As explained above, the fact that natural Schwann cells must becollected from peripheral nerves presents a difficulty for applicationto humans. Bone marrow stromal cells, on the other hand, are easy toobtain without damaging the human body. Moreover, since the cells have ahigh rate of growth and can therefore be supplied rapidly in largeamounts, the present invention makes possible a wider application ofbone marrow stromal cells for a variety of nervous system disorders.

[0094] Another major advantage afforded by bone marrow stromal cells istheir suitability for autologous transplantation. Collecting one's ownbone marrow stromal cells, inducing them to differentiate andtransplanting the differentiated cells into nerves produces no rejectionreaction and therefore requires no immunosuppressants or the like, whichshould allow regeneration to be achieved in a more stable manner. Sincebone marrow stromal cells can also be obtained from bone marrow banks,this method is also practical from the standpoint of supply.

[0095] As will be apparent by the examples provided below, the bonemarrow stromal cell-derived Schwann cells of the present invention areconsidered widely applicable for regeneration of peripheral nerves orcentral nerves. According to one mode, therefore, the invention providesthe bone marrow stromal cell-derived Schwann cells themselves. Due totheir different induced differentiation histories as mentioned above,they are artificially modified cells which are distinct from naturalSchwann cells. According to another mode, the invention provides thebone marrow stromal cell-derived Schwann cells in the form of apharmaceutical composition for neural regeneration. The bone marrowstromal cell-derived Schwann cells of the invention are suitable forautologous transplantation as explained above, but they may also beallogenically transplanted. This is because the cells of the nervoussystem are not as susceptible to immune system attack, and rejectionreaction can therefore be avoided by using donor cells with matchinghistocompatibility antigens from a bone marrow bank. The pharmaceuticalcomposition may also contain common pharmaceutically acceptablecarriers, buffers, salts, excipients and the like. The composition maybe injected into the affected site directly or it may be filled into ahollow tube for transplantation at the site of a severed central orperipheral nerve.

[0096] Although peripheral nerves intrinsically are capable ofregeneration, it is known that they cannot regenerate over gaps ofseveral centimeters; such cases are also considered to be included amongthe practical applications to peripheral nerves.

[0097] Central nerve conditions wherein reconstruction is consideredimpossible encompass a wide gamut of different conditions, includinginjury-related spinal cord damage or cerebrovascular damage and diseasesranging from blinding glaucoma to degenerative diseases such asParkinson's, which have a high estimated incidence rate among thepopulation. The pharmaceutical composition of the invention may be usedfor regeneration of many and various types of central nerves. Researchon methods of neural regeneration for the aforementioned conditions isan urgent social need, and the present invention is believed to havedirect application for the human body.

[0098] The invention will now be explained in greater detail through thefollowing examples, with the understanding that the examples are in noway limitative on the scope of the invention.

EXAMPLES Example 1 Induced Differentiation of Bone Marrow Stromal Cellsto Bone Marrow Stromal Cell-Derived Schwann Cells

[0099]FIG. 11 is a flow chart summary of the treatment process forinducing differentiation.

[0100] Stromal cells were extracted from the bone marrow of adult rats(wistar rats) and cultured. The culture medium used was MinimumEssential Medium Eagle Alpha Modification supplemented with 20% fetalcalf serum. After subculturing to four generations to reach 50%confluency, β-mercaptoethanol was added to a 1 mM concentration to theculture solution for a period of 24 hours. The medium was then exchangedwith medium containing 35 ng/ml retinoic acid. The latter culture mediumwas also Minimum Essential Medium Eagle Alpha Modification supplementedwith 20% fetal calf serum. After 3 days, the culture medium was againexchanged with medium containing 5 μM forskolin, 5 ng/mlplatelet-derived growth factor-AA, 10 ng/ml basic fibroblast growthfactor and 200 ng/ml Heregulin™. The cells were immunostained after 7days. Based on reaction with antibodies for P75, O4, S-100, GFAP andnestin, the cells exhibited reactivities equivalent to natural Schwanncells (see FIG. 8). The bone marrow stromal cells had been induced to bemorphologically similar to natural Schwann cells (see FIG. 9).

Example 2 Regeneration of Central Nerve (Severed Optic Nerve)

[0101] The cells obtained in Example 1 were collected by trypsintreatment and combined with mouse EHS tumor-derived Matrigelextracellular matrix (40234A, Collaborative Biomedical Products), andwere then transplanted after being packed into artificial tubes(HIP10-43 Hollow fiber cartridge, Amicon) which were then sutured tosevered optic nerves (central nervous system) of adult rats (Wistar).

[0102]FIG. 12 shows the results with GAP43 (Growth AssociatedProtein-43, a protein expressed during growth and elongation of nervefibers) labeling, 10 days after transplantation. The blue arrowsindicate the graft origins and the red arrows indicate the regeneratingfiber tips. Significant elongation of nerve fibers is seen with thedifferentiation-induced bone marrow stromal cells (right) compared tothe untreated bone marrow stromal cells (left). The nerve fiberelongation distance and number of fibers increased with the number ofweeks.

[0103] The results at the third week after grafting are shown in FIG.13. Here, FITC shown in green is the immunohistological detection ofanterograde labeled fibers obtained by injecting choleratoxin subunit Binto the vitreous body in order to specifically label only the opticnerve fibers, and it shows regeneration of nerve fibers in theartificial tubes. TexRed shown in red represents the cultured bonemarrow stromal cells pre-labeled with Brd-U. Alexa663 shown in bluerepresents MAG (Myelin-Associated Glycoprotein). Regeneration of theoptic nerves was observed in the artificial tubes packed with bonemarrow cells, and upon contacting these with the Brd-U labeled bonemarrow stromal cells, formation of myelin was confirmed.

Example 3 Regeneration of Peripheral Nerve (Sciatic Nerve)

[0104] The sciatic nerves of adult rats (Wistar rats) were severed andinoculated with artificial tubes packed with differentiation-inducedbone marrow stromal cells.

[0105]FIG. 14 shows the results with GAP43 (Growth AssociatedProtein-43) labeling, 7 days after transplantation. The blue arrowsindicate the graft origins and the red arrows indicate the regeneratingfiber tips. The nerve fibers are seen to be elongated with thedifferentiation-induced bone marrow stromal cells (right) compared tothe Matrigel (extracellular matrix) alone (left). The nerve fiberelongation distance and number of fibers increased with the number ofweeks.

[0106] The results at the fourth week after grafting are shown in FIG.15. Here, GFP (Green Fluorescent Protein) shown in green represents thebone marrow stromal cells illuminated by introducing the greenfluorescent protein gene (GFP) using a retrovirus, MAG shown in bluerepresents myelin protein, and Neurofilament shown in red representsregenerated nerve fibers. FIG. 15 clearly shows excellent regenerationof the sciatic nerve by the fourth week after transplantation.

[0107]FIG. 16, FIG. 17 and FIG. 18 show the results of regeneration ofsciatic nerves by the fourth week after transplantation. GFP shown ingreen represents bone marrow stromal cells illuminated by introductionof the green fluorescent protein gene, MAG shown in blue representsdetection of the myelin protein MAG using the fluorescent markerAlexa633, and Neurofilament shown in red represents regenerated nervefibers detected using the red fluorescent marker Alexa546. FIGS. 16 to18 clearly indicate total regeneration of the sciatic nerves (peripheralnerves) by the fourth week after transplantation. That is, it is seenthat the regenerated fibers (neurofilament) contacted with the bonemarrow stromal cells illuminated green with GFP, and that the bonemarrow stromal cells expressed the myelin protein MAG to form myelin.

REFERENCES

[0108] 1. Schwab, M E et al: Rat CNS myelin and a subtype ofoligodendrocytes in culture represent a nonpermissive substrate forneurite growth and fibroblast spreading. J Neurosci 8: 2381-2393, 1988

[0109] 2. Hall, S et al: Electron microscopic study of the interactionof axons and glia at the site of anastomosis between the optic nerve andcellular or acellular sciatic nerve grafts. J Neurocytol 18: 171-184,1989

[0110] 3. Snow, D M et al: Sulfated proteoglycans in astroglial barriersinhibit neurite out-growth in vitro. Exp Neurobiol 109: 111-130, 1990

[0111] 4. Blaugrund, E et al: Disappearance of astrocytes and invasionof macrophages following crush injury of adult rodent optic nerves:implications for regeneration. Exp Neurol 118: 105-115, 1992

[0112] 5. Bastmeyer, M et al: Similarities and differences between fisholigodendrocytes and Schwann cells in vitro. Glia 11: 300-314, 1994

[0113] 6. Vidal-Sanz, M et al: Axonal regeneration and synapse formationin the superior colliculus by retinal ganglion cells in the adult rat. JNeurosci 7: 2894-2909, 1987

[0114] 7. Dezawa, M et al: The role of Schwann cells during retinalganglion cell regeneration induced by peripheral nerve transplantation.Invest Ophthalmol Vis Sci 38: 1401-1410, 1997

[0115] 8. Aguago, A. J. et al.; A potential for axonal regeneration inneurons of the adult mammalian nervous system. In Nervous SystemRegeneration, B. Haber, J. R. Perez-Polo, G. A. Hashim and A. M. G.Stella eds., pp.327-340, Alan R. Liss, New York, 1983

[0116] 9. Chen, M. S. et al.; Nogo-A is a myelin-associated neuriteoutgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature403: 434-439, 2000

[0117] 10. Taito, M., professor, Tohoku University), “Studies inestablishing differentiation function-maintaining cell lines and usingthem for reconstruction of biohistological function”, NEDO 1999 TeianKobo Jigyo Seika Hokoku: 97S09-003.

1. A method of inducing bone marrow stromal cells to differentiate intobone marrow stromal cell-derived Schwann cells in vitro, comprising thesteps of: (1) collecting bone marrow stromal cells from bone marrow, andculturing said cells in a standard essential culture medium supplementedwith a serum; (2) adding a reducing agent to said culture medium, andfurther culturing said cells; (3) adding a differentiation inducingagent to said culture medium, and further culturing said cells; and (4)adding a cyclic AMP-augmenting agent or a cyclic AMP analogue, and/or adifferentiation, survival and growth stimulating factor which acts onnerves and glial cells to said culture medium, and further culturingsaid cells to obtain said bone marrow stromal cell-derived Schwanncells.
 2. The method as defined in claim 1, wherein said standardessential culture medium is an Eagle's alpha modified minimum essentialmedium.
 3. The method as defined in claim 1, wherein said serum is fetalcalf serum.
 4. The method as defined in claim 1, wherein said reducingagent is an SH reagent.
 5. The method as defined in claim 4, whereinsaid SH reagent is β-mercaptoethanol.
 6. The method as defined in claim1, wherein the concentration of said reducing agent is between 1 nM and10 mM.
 7. The method as defined in claim 1, wherein the culturing timein step (2) is between 1 hour and 5 days.
 8. The method as defined inclaim 1, wherein said differentiation inducing agent is retinoic acid.9. The method as defined in claim 1, wherein the concentration of saiddifferentiation inducing agent is between 0.001 ng/ml and 1 μg/ml. 10.The method as defined in claim 1, wherein the culturing time in step (3)is within 30 days.
 11. The method as defined in claim 1, wherein saidcyclic AMP-augmenting agent or cyclic AMP analogue is forskolin.
 12. Themethod as defined in claim 1, wherein the concentration of said cyclicAMP-augmenting agent or cyclic AMP analogue is between 0.001 ng/ml and100 μg/ml.
 13. The method as defined in claim 1, wherein said glial celldifferentiation and survival stimulating factor is selected from thegroup consisting of neuregulin, platelet-derived growth factor-AA, basicfibroblast growth factor, and mixtures thereof.
 14. The method asdefined in claim 13, wherein said neuregulin is Heregulin, a subtype ofthe same.
 15. The method as defined in claim 1, wherein theconcentration of said glial cell differentiation and survivalstimulating factor is between 0.001 ng/ml and 100 μg/ml.
 16. The methodas defined in claim 1, wherein the culturing time in step (4) is within30 days.
 17. Bone marrow stromal cell-derived Schwann cells obtained bythe method as defined in any one of claims 1 to
 16. 18. Bone marrowstromal cell-derived Schwann cells.
 19. A pharmaceutical composition forneural regeneration, comprising bone marrow stromal cell-derived Schwanncells as defined in claim 17 or
 18. 20. A method of treating neuraldisease by transplanting Schwann cells as defined in claim 17 or 18 or apharmaceutical composition as defined in claim 19 into a patient withneural disease to promote regeneration of the neural cells with whichthe disease is associated.